Euro-Collins Solution Versus Uw-Solution for Long-Term Liver Preservation in the Isolated Rat-Liver Perfusion Model

To compare UW-solution (UW) and Euro-Collins (EC) for long-term liver preservation we investigated the morphology and metabolic capacity of rat liver after 18 and 42-hours cold-storage in either UW or EC. After harvesting the rat liver was transferred to a perfusion chamber where it was perfused for 10 min with UW or EC at 4°C. Thereafter livers were stored at 4°C in UW or EC for 18 hours (both groups n = 6) or for 42 hours (both groups n = 8). After 18-hr or 42-hr cold-storage a 2-hr warm perfusion (37°C) was started with Krebs-Ringer solution with carbogen to which 125Iodine-triiodothyronine (T3) was added. Control livers (n = 8) were immediately perfused with Krebs-Ringer without cold-storage. The following parameters were assessed: ASAT-levels in the perfusate, T3-metabolites in the bile and the perfusate, the perfusion pressure, the volume of bile secreted and light-microscopical morphology at the end of the warm perfusion period. After cold storage in UW-solution the ASAT-levels in the perfusate were lower than after storage in EC as well as the perfusion pressures. These livers demonstrated a better T3-metabolism and secreted more bile than EC-stored livers. Histological examination showed more tissue damage in the EC-stored livers than in the UW stored livers. We conclude that cold-storage of rat liver in UW-solution resulted in a better morphology and metabolic capacity as compared with EC-solution.


INTRODUCTION
Until Belzer and co-workers introduced a new solution for preservation of solid organs the preservation time for human liver was limited to 6-10 hours using a modified version of a solution introduced by Collins in 19692. This modified version is called Euro-Collins solution and it was used in most countries in clinical kidney, liver and pancreas transplantation3.
Preservation of rat liver after flushing with Euro-Collins and cold-storage in this medium for longer than 12 hours resulted in irreversible damage to sinusoidal cells and hepatocytes4. Many techniques have been investigated with the aim to prolong the preservation period: prostacyclin infusion before ischemia5'7, rapid flush methods8, rapid cooling of the organ and the use of different storage solutions1 '19. By prolongation of the preservation period histocompatibility matching between the donor and the recipient becomes possible, the donor pool increases by enlarging the area from which donor livers can be obtained and liver transplantation becomes a semi-elective procedure instead of an emergency one.
Recently successful 24-hours-preservation of the liver using UW-solution, has been reported1. The aim of our study was to investigate the morphology and metabolic capacity of rat liver after 18 and 42 hours cold storage in UW-solution (UW) as compared with cold storage in Euro-Collins solution (EC).

MATERIAL AND METHODS
Male Wistar rats, weighing 200-300 g, were randomly allocated to four groups: 18-hr storage UW solution (n 6), 18-hr EC (n 6), 42-hr UW (n 8) and 42-hr EC (n 8). Eight other rats were used as controls (no preservation). All rats were deprived of chow 48 hours prior to the isolation of the liver but had free access to water.

Surgical Procedures
Anesthesia was induced by intraperitoneal injection of pentobarbital sodium (60 mg/kg body weight). The surgical and recirculating perfusion technique were as described earlier2. The perfusion medium was gassed with carbogen (95% O2, 5% CO2), 600 mL/min. After harvesting, the liver was transferred to the perfusion cabinet where it was perfused for 10 minutes with either one of the two solutions (UW or EC) at 4C (Figure 1). After this preperfusion period the liver was stored at 4C in 100 ml of either one of the preservation solutions for 18 or 24 hours. After this cold storage period the liver was placed back in the cabinet and the second perfusion period started with a 1 hour-period at 37C with Krebs-Ringer solution. Livers of the control rats were used immediately after isolation without coldstorage. After adding 2nM L-triiodothyronine and 125Iodine-triiodothyronine (7 106 counts) to the perfusion medium, the perfusion was continued for another hour (experimental period). Liver Tests Function of the liver was evaluated by several parameters: during the first hour of perfusion ASAT-activity was determined 10 and 55 minutes after starting; the perfusion pressure as determined by the height of the perfusion column (to obtain a medium flow of 40 mL/min through the liver); the amount of bile secretion, which was measured every 10 min during the experimental period and histological changes in a wedge biopsy of liver tissue taken at the end of the experiment. Histological evaluation was performed by an observer who had no knowledge of the kind of storage solution that was used. Histological examination concerned the architecture of the whole liver tissue specimen as well as degenerative changes of the hepatocytes, the cohesion between the hepatocytes, changes in cytoplasm and nucleus, the appearance of the portal fields (bile ducts, edema, inflammatory infiltration, bleeding, etc.) and the amount of glycogen stored in the cells.

Triiodothyronine-kinetics
To evaluate the metabolic capacity of the hepatocytes, triiodothyronine-kinetics were studied. Two components were investigated" transport of triiodothyronine over the hepatocyte cell membrane and the deiodination and conjugation of triiodothyronine. These two phases of thyroid hormone metabolism are both energy-consuming processes and are sensitive parameters of liver function2 '22. Aliquots of the perfusion medium were taken 0.5 min after the start of the experimental period and at one minute intervals until 10 min after start of the experimental period, then at 5-10 min intervals until one hour after the start. Perfusate and bile samples were stored at -20C until further analysis. All samples were analysed by chromatography on small Sephadex LH-20 columns equilibrated with 0.1 M HC1. Iodide was eluted with HCI; triiodothyronine-glucuronide, triiodothyronine-sulfate and triiodothyronine with sodium-acetate, H20 and NaOH/ethanol (50/50 vol/vol) respectively.Fractions of 1 ml were collected and counted for radioactivity. For analysis of triiodothyronine-kinetics the tracer triiodothyronine disappearance curve from medium was fitted into a two pool model developed by Doctor et al. 23. This model enables discrimination between triiodothyronine-transport and triiodothyronine-metabolism in general in the rat liver.
Statistical evaluation of the experiments was performed by the Mann-Whitney U-test. Results are presented as mean +_ standard error of the mean.

Macroscopic Appeara'nce
At the end of the experiments there were slight macroscopic differences between the four cold-storage groups of livers. All livers stored in UW were less pale and showed fewer white ischemic spots than livers stored in Euro-Collins. The differences between the groups were more pronounced after 42-hr than after 18-hr coldstorage.
ASA T-activity In the UW-stored group (18 and 42-hr) ASAT-levels were significantly lower at 10 and 55 min (in the first hour of perfusion) as compared with EC-stored (18 and 42hr) livers. In the control group ASAT-levels at 10 min were lower than in the 42-hr EC group, whereas at 55 min ASAT-levels in the control group were lower than in the UW (42-hr) and in both EC groups (Table 1.). Table 1

Histology
In general UW-stored livers had less histological changes than EC-stored livers, in the 18-hr as well as in the 42-hr cold storage groups. In 2/6 EC (18-hr), in 0/6 UW (18-hr), in 5/8 EC (42-hr) and in 1/8 UW (42-hr)-livers more than 10% of the hepatocytes were necrotic. None of the livers in the control group showed more than 10% necrosis. Edema in the portal triads was less evident in UW-stored (18 and 42-hr) livers. Degenerative changes of the liver-parenchyma (such as diminished cohesion with rounded hepatocytes, pale vacuolated cytoplasm with eosinophilic changes, pycnosis of nuclei and nuclear remnants in Kupffer-cells and in sinusoidal vessels) were less pronounced in UW-stored (18 and 42-hr) livers as compared with EC-stored (18 and 42-hr) livers. Livers in the control group had a nearly normal histological appearance with only a few hepatocytes showing degenerative changes. There were no evident differences in glycogen content in the hepatocytes, which was diminished in the control, the UW (18-hr) and the EC (18hr) group. In the UW (42-hr) and the EC (42-hr) group the amount of glycogen was severely diminished. In two thirds of the livers in both 42-hr cold-storage groups bacterial colonies were found, whereas in the control, the UW (18-hr) and the EC (18-hr) group no bacterial colonies were observed.

DISCUSSION
The aim of our study was to evaluate two cold storage solutions, the Euro-Collins-solution and the newer UW-solution in the isolated rat liver perfusion model. The isolated liver perfusion model is a good alternative to studies in which the liver is grafted for assessment of preservation solutions11. In transplantation experiments immunological and technical problems may obscure the overall results. The isolated rat liver model is a simple, rapid and inexpensive way to assess liver function without interference from other organ systems in the body, while the integrity of the liver is not affected. ASAT-levels in the perfusate differed significantly between the 18-hr coldstorage groups, and between the 42-hr cold-storage groups, but showed considerable intra-group variability. The latter may be due to continuous perfusion with the same perfusate which can cause accumulation to the effects of these values. Other groups have also found great variation in ASAT-levels and stated that this parameter in the perfusate was not related to later liver function in htlmans 24 or in rabbits1.
More sophisticated methods to monitor liver function are available nowadays, one of these is assessment of thyroid hormone metabolism. Thyroid hormone transport and intracellular-metabolism are energy consuming processes and therefore dependent on the availability of energy stores21'22'2526. It has been shown that in conditions of reduced ATP-availability (in primary rathepatocyte cell cultures) both triiodothyronine transport and metabolism are decreased23. Our results showed a similarly decreased transport of triiodothyronine after cold storage in both UW and EC groups, whereas triiodothyronine-metabolism is more diminished after 42-hr cold-storage in either EC or UW than after 18-hr cold-storage in these solutions, indicating that triiodothyronine-metabolism is a valuable monitor of the metabolising capacity of the liver.
The perfusion pressure is a reflection of the amount of tissue edema. The perfusion pressure in UW (42-hr) stored livers was significantly lower than that of livers stored in EC (42-hr), whereas the perfusion pressure in the UW (18-hr) and in the EC (18-hr) groups did not differ. This finding is consistent with Belzer's concept of adding raffinose and hydroxyethyl starch to the cold-storage solution to prevent expansion of the extracellular space28. As other groups have also asserted we confirm that bile production is a simple and reliable monitor of liver function11'24'9-31. When we compare bile production with perfusion pressure and triiodothyronine-kinetics, the differences between the groups are very consistent.
Because of the high metabolic rate, hepatic cells are most sensitive to the deleterious effects of anoxia3:. Therefore light microscopic examination of liver tissue (biopsies) has proved to be a good monitor of ischaemic damage to the cells4'14 '19'33'34. Others have found light microscopic investigation to be of limited value35. In our study we found a good correlation between the histological appearance and the function of the hepatocytes. The amount of glycogen in the hepatocytes was not a reliable monitor of liver condition after 42-hr cold-storage. The decreased amount of glycogen could also be due to the 48-hr fasting period preceding the cold-storage period, because hepatocytes in the control group and in the 18-hr cold storage groups also contained less glycogen.
Summarising these results we conclude that in the isolated rat liver perfusion model cold-storage with UW-solution gave better metabolic performance and morphological results than with Euro-Collins solution. The differences between UW-solution and Euro-Collins solution are more pronounced after 42-hr than after 18-hr cold storage.